小鼠角膜γδT细胞去除效率的双光子活体动态监测

Dynamic monitoring of γδT cells in murine cornea in vivo using two-photon laser scanning microscopy

目的比较抗体中和后角膜与耳部皮肤γδT细胞的去除效率,探寻一种能够活体动态监测角膜γδT细胞数量变化的替代方法。方法应用双光子激光扫描显微镜对抗体中和前后小鼠耳部皮肤γδT细胞数量进行监测,同时于抗体中和后6 h、12 h、24 h对小鼠角膜进行取材染色,计数角膜中的γδT细胞,比较两者去除效率是否一致。结果正常小鼠角膜的γδT细胞常分布于角膜缘上皮及浅基质层,上皮层的γδT细胞形态不规则,常伴有突起,基质层γδT细胞多呈圆形或椭圆形,细胞数量为(27±4)个。抗体中和后,小鼠角膜γδT细胞数量逐渐减少,6 h、12 h和24 h细胞数量均明显少于去除前(P=0.03、0.00、0.00),去除效率分别为48%、78%、96%。正常小鼠耳部皮肤γδT细胞呈树突状,均匀分布于皮肤表皮层,细胞数量为(60±9)个,抗体中和后6 h、12 h和24 h,耳部皮肤γδT细胞数量与去除前相比亦明显减少(P=0.00、0.00、0.00),去除效率分别为43%、72%、95%。结论抗体中和后角膜与耳部皮肤γδT细胞数量均逐渐下降,两者去除效率随时间变化一致,监测小鼠耳部皮肤γδT细胞是动态监测角膜γδT细胞数量变化的理想替代方法。

Objective To compare the removal efficiency of γδT cells between cornea and ear skin and develop an alternative method for dynamic monitoring of γδT cells in mouse cornea in vivo using 2-photon laser scanning microscopy. Methods The γδT cells in mouse ear skin were monitored before and after antibody neutralization,and the mice corneas were excised and stained for counting γδT cells at 6 h,12 h,24 h after antibody neutralization by using 2-photon laser scanning microscopy,follow ed by comparison of the removal efficiency of γδT cells betw een the cornea and ear skin. Results The γδT cells in normal mouse cornea were often distributed in the limbal epithelium and superficial stromal layer. The irregular morphology of γδT cells in the epithelial layer was often accompanied by protuberances,while the stromal γδT cells were mostly round or oval and the number of cells was approximately 27 ± 4. After antibody neutralization,the number of γδT cells in the cornea of mice gradually decreased,and the number of cells at 6 h,12 h and 24 h was significantly low er than that of before depletion（ P = 0. 03,0. 00,0.00）,and the removal efficiencies were 48%,78%,and 96%,respectively. The γδT cells in ear skin of the normal mice were ellipse or stellate with cell processes and they were located in epidermal layer,and the cell number was about 60 ± 9. After antibody neutralization,the number ofγδT cells were significantly reduced at 6 h,12 h and 24 h compared with before depletion（ P =0. 000,0. 000,0. 000） and the removal efficiency were 43%,72% and 95%,respectively. Conclusion The number of γδT cells in the cornea and ear skin is gradually decreased after antibody neutralization,and their removal efficiency is consistent with time. Therefore,monitoring theγδT cells in the mouse ear skin is an ideal alternative to dynamically monitoring the changes in the number of γδT cells in the cornea in vivo.